Modified (80-113) beta interferons

ABSTRACT

Modified beta interferons containing amino acid substitutions in the beta interferon amino acids 80 to 113 are described. These modified beta interferons exhibit changes in the antiviral, cell growth regulatory or immunomodulatory activities when compared with unmodified beta interferon.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention describes the use of recombinant DNA technology for thedesign and synthesis of novel, modified interferons. More specificallythe invention relates to interferons not known in nature which areintended for use in viral and neoplastic diseases, and immunosuppressedand immunodeficient conditions.

2. Description of the Prior Art

The interferons are a class of proteins that occur in vertebrates andact as biological regulators of cell function which include increasingresistance to pathogens, limiting cell growth and modulating the immunesystem. The most studied property of the interferons is their ability toconvert cells into an "antiviral state" during which they are moreresistant to virus replication (Lengyel, Annual Review of Biochemistry,51, 251, 1982). In addition to conferring antiviral resistance to targetcells, interferon (IFNs) have antiproliferative (antigrowth) properties(Stewart, 1979, The Interferon System, Springer Berlin) It has clearybeen shown that interferons produced naturally act as antiviral andantiproliferative agents (Gresser et al, Biochim. Biophys. Acts, 516,231, 1978; J. Exp Med, 144, 1316, 1976).

The IFNs by virtue of their antigenic biological and physico-chemicalproperties, may be divided into three classes: type I, IFN-α("Leucocyte") and IFN-β ("fibroblast"); and type II IFN-γ ("immune")(Stewart et al, Nature, 286, 10, 1980). Both genomic DNA and cDNA clonesof type I and type II IFNs have been isolated and sequenced, and thepotential protein sequences deduced (e.q. Pestka, Arch. Biochem.Biophys., 221, 1, 1983). Whilst in man only one IFN-β and IFN-γ gene areknown, human IFN-α is specified by a multigene family comprising atleast 20 genes. The classification of IFN-β and IFN-α as type Iinterferons is in part determined by their significant degree ofhomology, >23% at the protein level (Taniquchi et al, Nature, 285, 547.1980).

Whilst the mechanism of action of interferons is not completelyunderstood, certain physiological or enzymatic activities respond to thepresence of the interferons. These activities include RNA and proteinsynthesis. Among the enzymes induced by interferons is (2'-5') (A)nsynthetase which generates 2'-5' linked oligonucleotides, and these inturn activate a latent endoribonuclease, RNAse L, which cleavessingle-strand RNA, such as messenger RNA (mRNA) and ribosomal RNA(rRNA). Also induced by IFNs is a protein kinase that phosphorylates atleast one peptide chain initiation factor and this inhibits proteinsynthesis (Lengyel, ibid. p. 253) IFNs have been shown to be negativegrowth regulators for cells by regulation of the (2'-5') An synthetaseactivity (Creasey et al, Mol. and Cell Biol, 3, 780, 1983). IFN-β wasindirectly shown to be involved in the normal regulation of the cellcycle in the absence of inducers through the use ofanti-IFN-β-antibodies. Similarly, IFNs have been shown to have a role indifferentiation (Dolei et al, J. Gen. Virol., 46, 227, 1980) and inimmunomodulation (Gresser, Cell. Immunol., 34, 406, 1977). Finally, IFNsmay alter the methylation pattern of mRNAs and alter the proportion offatty acids in membrane phospholipids, thereby changing the ridigity ofcell membranes.

These and other mechanisms may respond to interferon-like molecules invarying degrees depending on the structure of the interferon-likepolypeptide. Preliminary evidence (U.K. Pat. No. GB 2 090 258A) suggeststhat members of the multigene IFN-α family vary in the extend andspecificity of their antiviral activity (Pestka ibid.). For example,combination of IFN-αA with IFN-αD resulted in "hybrid" genes which showantiviral properties that are distinct from either parent molecule (Wecket al, Nucl. Acids Res., 9, 6153, 1981; De La Maza et al, J. IFN Res.,3, 359, 1983; Fish et al, Biochem. Biophys. Res. Commun., 112, 537,1983; Weck et al, Infect Immun., 35, 660, 1982). However, hybrid humanIFNs with significantly increased human cell activity/specificity havenot yet been developed. One patent has been published describing IFN-β/αhydrids (PCT/U.S. No. 83/00077). This patent described three examples,none of which have significantly improved activity. The three exampleswere constructed using two naturally occurring restriction sites. Theresulting hybrid inteferons were (1) alpha 1 (1-73)-beta (74-166); (2 )beta (1-73)-alpha 1 (74-166); and (3) alpha 61A (1-41)-beta (43-166).These three examples differ structurally from the examples of thepresent invention. These three examples were based upon the accidentallocation of two restriction sites and not upon the intentionallydesigned DNA and amino acid sequences of the present invention.

It is envisaged that a modified interferon will display a newadvantageous phenotype. The design and synthesis of new interferon-likepolypeptides composed of portions of IFN-β and other amino acidsequences is advantageous for the following reasons:

1. New IFNs can be created which show a greater antiproliferative toantiviral activity (and vice versa) resulting from the selectiveactivation of only some of the normal interferon-induced biochemicalpathways.

2. The affinity of hybrid or modified IFNs for cell surface receptorscan differ from that of naturally occurring interferons. This will allowselective or differential targeting of interferons to a particular celltype, of increased affinity for the receptor--leading to increasedpotency against a particular virus disease or malignancy.

3. It will be possible to design novel IFNs which have an increasedtherapeutic index, thus excluding some of the undesirable side effectsof natural IFNs which limit their use (Powledge, T.M., Biotechnology, 2,214, March 1984).

4. Novel IFNs can include in the design structures which allow increasedstability to proteolytic breakdown during microbial synthesis.

5. Novel IFNs can be designed to increase their solubility or stabilityin vivo, and prevent non-specific hydrophobic interactions with cellsand tissues.

6. Novel IFNs can be designed which are more readily recovered from themicrobial supernatant or extract and more easily purified.

Additional Relevant Patent Applications

U.K. No. GB 2 116 566A--Animal interferons and processes for theirproduction.

U.S. No. 4,414,150--Hybrid human leukocyte interferons

U.K. No. GB 2 068 970A--Recombinant DNA technique for the Preparation ofa protein resembing human interferon.

SUMMARY OF THE INVENTION

Recombinant DNA technologies were successfully applied to producemodified beta interferon-like polypeptides, nucleic acids (either DNA orRNA) which code for these modified beta interferons, plasmids containingthe DNA coding for the modified beta interferons and procedures for thesynthesis of these modified beta interferons. Each of the amino acids80-113 of human beta interferon may individually be replaced by anyother amino acid. This replacement may be accomplished in groups of fourto thirty-three amino acids. One preferred embodiment is the replacementof four to twenty-four of the amino acids 82 to 105 of human betainterferon by four to twenty-four other amino acids Another preferredembodiment is the replacement of beta interferon amino acids 103-112 byfour to ten other amino acids.

The beta interferon amino acids 103-112 may be replaced sequentially bycorresponding human alpha interferon amino acids. Correspondence isdefined by usage in this invention. Among the alpha interferons arealpha 1, alpha 2 and alpha H. The alpha and beta interferons from anymammal may be used including but not limited to humans or otherprimates, horses, cattle, sheep, rabbits, rats, and mice. In oneembodiment of the invention, the leucine occurring at position 84 inhuman beta interferon may optionally be replaced by proline. Yet anotherembodiment of the invention discloses the use of the modified betainterferons where in one or more of the antiviral, cell growthregulatory, or immunomodulatory activities is substantially changed fromthat of the unmodified beta interferon. Particularly preferredembodiments are the amino acid sequences of IFNX405, 423, and 429. Yetanother preferred embodiment of the invention is DNA or RNA sequenceswhich code for the synthesis of IFNX405, 424, or 429. Still anotherembodiment is a plasmid or a cell containing a DNA sequence capable ofcoding for the synthesis of IFNX405, 423, or 429. Yet another embodimentof the invention is a pharmaceutical composition continuing an efectiveamount of IFNX405, 423, or 429. A final embodiment of the invention isthe use of pharamaceutical compositions containing the modified betainterferons in a method of treating viral infections, regulating cellgrowth or regulating the immume system.

The modified interferons have altered activities measurable in vitrosuch as antiviral or immunomodulatory activity. Target cell specificitycan also be altered.

An increased target cell specificity can result in an improvedtherapeutic index. This should exclude some of the side effects causedby the use in humans of naturally occurring IFNs.

This invention relates to the production in sufficient amounts of novel,highly specific interferon-like molecules suitable for the prophylacticor therapeutic treatment of humans--notably for viral infections,malignancies, and immunosuppressed or immunodeficient conditions.

BRIEF DESCRIPTION OF THE CHARTS AND TABLES

FIG. 1 shows the Sternberg-Cohen 3D model of α₁ and β interferons (Int.J. Biol. Macromol, 4, 137, 1982).

Chart 2 (a to c) shows the ligated oligonucleotides used in theconstruction of the novel, modified IFNs.

Chart 3 (a to d) shows the complete necleotide sequences of the novel,modified IFn genes and the encoded amino acid sequences.

Chart 4 shows the nucleotide sequence of the trp promoter used toinitiate transcription of the novel, modified IFN genes.

Table 1 compares expression, antiviral and antiproliferative activityfor IFNX423, IFNX429, and IFN-β present in crude bacterial extracts.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Introduction

The IFN-β gene is a unique gene but shows some significant homologies tothe multigenic IFN-α family (Rubinstein, Biochim. Biophys. Acta, 695, 5,1982). Sternberg and Cohen (Int. J. Biol. Macromol., 4, 137, 1982) haveproposed a similar secondary structure for IFN-β and IFN-α₁. Structureprediction studies suggest four α-helices which can be "packed" into aright handed bundle (FIG. 1) similar to that observed in severalunrelated protein structures as determined by X-ray crystallography. Thedesign of some of the modified interferons described herein is derivedfrom our interpretation of the Sternberg/Cohen mode. Since IFNs β and αare believed to bind to the same receptor at the cell surface it ispossible to introduce variability into IFN-β by replacing specific areaswith IFN-α segments.

The field of the present invention is the design, synthesis andcharacterization of interferon-like molecules related to IFN-β which mayhave sequences between amino acids residues 85-115 replaced with anyother amino acid sequence, unrelated protein sequences, or sequencessimilar to those of IFN-α's, IFN-β's or IFN-γ found in mammals and othervertebrates.

Each amino acid in the 80 to 113 region can be replaced by any othernaturally occurring amino acids. The naturally occurring amino acids andtheir nomenclature are: alanine (Ala or A); valine (Val or V); leucine(Leu or L); isoleucine (Ile or I); proline (Pro or P); phenylalanine(Phe or F); tryptophan (Trp or W); methionine (Met or M); glycine (Glyor G); serine (Ser or S); threonine (Thr or T); cysteine (Cys or C);tyrosine (Tyr or Y); asparagine (Asn or N); glutamine (Glu or Q);aspartic acid (Asp or D); glutamic acid (Glu or E); lysine (Lys or K);arginine (Arg or R); and histidine (His or H).

Though binding of hybrid IFN-β's (α₁ and α₂ in Streuli et al, Proc.Natl. Acad. Sci. USA, 78, 2848, 1981), an attempt was made to analysethe number and nature of idiotypes involved in the receptor binding siteof IFN-α's. Two sites were proposed as constituting the binding site,one in the amino-terminal half and the other in the carboxy-terminalhalf of IFN-α. The two major regions of partial homology between IFN-α'sthe IFN-β occur between amino acid residues 28-80 and 115-151 which maywell correspond to the above mentioned idiotypes. Evidence that the28-80 region may be important in receptor binding come from the findingthat polyclonal antibodies raised against a synthetic peptide composedof IFN-α₂ amino acids 24-81, bind to IFN-α₂ and prevent it interactingwith its cell receptor (Dreiding, TNO Interferon Meeting, Rotterdam,1983).

Little or no information is available on the function or importance ofthe region of IFN-β or IFN-α's between amino acid residues 82 and 115(IFN-β numbering), which includes a computer-predicted α-helical region(Sternberg and Cohen, Int. J. Biol. Macromol., 4, 137, 1982).

However, an amino terminal fragments of IFN-α₂ of 110 amino acids(Ackerman et al, Proc. Natl. Acad. Sci. USA, 81, 1045, 1984) retains asmall portion of its antiviral activity. C-terminal fragments are notactive.

The following are examples of novel, modified IFNs with amino acidreplacements in the 82-115 region of IFN-β to illustrate the invention,and are not intended to limit the scope of the invention in any way.Below are described techniques used in the design, chemical synthesisand insertion of DNA fragments in the 82-115 region of the human IFN-βgene. The resultant novel, modified IFNs are hereafter described asgroup III IFNs. Decreased antiviral and/or immunostimulating activityare among the altered properties shown by some of the group III novelIFNs with amino acid replacements in the 82-115 region. The techniquesdescribed will be familiar to anyone skilled in the art [see alsoMolecular Cloning, A Laboratory Manual, eds. Maniatis et al, Cold SpringHarbor Laboratories].

Design of the synthetic gene fragments

The nucleotide sequences of each synthetic DNA fragment (Charts 2a to c)were designed utilizing the following criteria:

1. Codon utilization (where it deviates from natural IFN-β genesequence) was optimized for expression in E.coli. Natural IFN-β genesequences were used as far as possible in order to obtain levels ofexpression of novel IFNs as high as that of IFN-β from plasmid pGC10(see Table 1). pGC10 (˜4,440 bp) expresses the natural IFN-β gene at ahigh level and is identical to p1/24 (Patent Application GB 2 068 970A,hereby incorporated by reference) except for the ribosome binding sitesequence shown in Chart 4 and the deletion of the ˜546 bp BglII-BamHIfragment.

2. Sequences which might anneal to each other in the assembly of thechemically synthesized fragment (Chart 2) were not included in thedesign (within the limits allowed by the redundancy in the geneticcode).

Chemical Synthesis of Gene Fragments

Oligodeoxyribonucleotides were synthesized by the phosphoramidite method(M. H. Caruthers in "Chemical and Enzymatic Synthesis of GeneFragments", ed. H. G. Gasen and A. Lang, Verlag Chemie, 1982, p. 71) oncontrolled pore glass (H>Koster et al, Tetrahedron, 40, 103, 10984).Fully protected 2'-deoxyribonucleotide 3'-phosphoramidites weresynthesized from the protected deoxyribonucleotide andchloro-N,N-(diisopropylamino)methoxyphosphine (L. J. McBride and M. H.Caruthers, Tetrahedron Lett., 24, 245, 1983 and S. A. Adams et al, J.Amer. Chem. Soc., 105, 661, 1983). Controlled pore glass supports weresynthesized as described (F. Chow et al, Nuc. Acids Res., 1981, 9, 2807)giving 30-50 μmol deoxynucleoside per gram.

The functionalised controlled pore glass (50 mg) was treated in asintered glass funnel at ambient temperature sequentially with:

1. Dichloromethane (3 ml, 10s)

2. 3% (v/v) dichloroacetic acid in dichloromethane (2 ml, 120s)

3. dichloromethane (3 ml, 10s)

4. anhydrous acetonitrile (3 ml, 10s)

5. phosphoramidite monomer (0.06M)/tetrazole (0.23M) in anhydrousacetonitrile (1 ml, 120s)

6. acetonitrile (3 ml, 10s)

7. dimethylaminopyridine (0.07M) in aceticanhydride/2,6-lutidine/acetonitrile (1/2.6 v/v) (1 ml, 60s)

8. acetonitrile (3 ml, 10s)

9. iodine (0.2M) in 2,6-lutidine/tetrahydrofuran/water (1/2/2 v/v) (1ml, 30s)

10 acetonitrile (3 ml, 10s)

The cycle was repeated with the appropriate phosphoramidite monomeruntil the immunogenetic chain was complete. The coupling efficiency ofeach cycle was monitored by spectrophotometric assay of the liberateddimethoxytrityl alcohol in 10% (w/v) trichloroaceticacid/dichloromethane at 504 nm. After completion of the synthesis, theprotecting groups were removed and the oligomer cleaved from the supportby sequential treatment with 3% (v/v) dichloroaceticacid/dichloromethane 9120s, thiophenol/triethylamine/dioxan (1/1/2 v/v)(1h) and concentrated ammonia at 70° C. (4h). The deprotectedoligonucleotides were purified either by HPLC on a Partisil 10 SAXcolumn using a gradient from 1M to 4M triethylammonium acetate pH 4.9 at50° C. or by electrophoresis on a denaturing 15% polyacrylamide gel (pH8.3).

Ligation of Oligonucleotide Blocks

500 pmole aliquots of the oligonucleotides were phosphorylated with 1unit of T4 induced polynucleotide kinase in 20 μl of a solutioncontaining 1000 Ci/pmole [³² p]γ-ATP (2.5 Ci/mMole), 100 μM sperimidine,20 mM DTT, 10 mM MgCl₂, 50 mM Tris-HCl (pH 9.0) and 0.1 mM EDTA for 60minutes at 37° C. The mixtures were then lyophilized and eacholigonucleotide purified in a denaturing 15% polyacrylamide gel (pH8.3). After elution from the gel, the recovery was determined bycounting the radioactivity.

Blocks (length 30-50 bases) were assembled by combining 25 pmole of eachphosphorylated component with equimolar amounts of the unphosphorylatedoligomers from the complementary strand. The mixtures were lyophilizedand then taken up in 15 μl water and 2 μl 10×ligase buffer (500 mMTris-HCl pH 7.6, 100 mM MgCl₂). The blocks were annealed at 100° C. for2 minutes, then slowly cooled to room temperature (20° C.). 2 μl 200 mMDTT and 0.5 μl 10 mM ATP were added to give final concentrations of 20mM DTT and 250 μM ATP in 20 μl. 1.25 units of T4 DNA ligase were alsoadded. After 18 hours at 20° C., the products were purified in a 15%polyacrylamide gel under denaturing conditions.

Two duplex blocks were then constructed from the single-stranded pieces.(These were 150 base pairs and 75 base pairs). 1.5 pmole of each blockwere taken and the mixtures lyophilized. Annealing was carried out in 15μl water and 2 μl 10×ligase buffer at 100° C. for 2 minutes, then slowlycooled to 10° C. 2 μl 200 mM DTT, 0.5 μl 10 mM ATP and 1.25 units T4 DNAligase were added. The reaction was left at 10° C. for 18 hours. Theproducts were then purified in a 10% native polyacrylamide gel.

The final product was assembled by combining 0.4 pmole of the twoduplexes. The mixture was lyophilized and then taken up in 15 μl waterand 2 μl 10×ligase buffer. It was annealed at 50° C. for 2 minutes andthen slowly cooled to 10° C. 2 μl 20 mM DTT, 0.5 μl 10 mM ATP and 1.25units ligase were then added and the reaction left at 10° C. for 18hours. The final product was purified in a 5% native polyacrylamide gel.After elution and ethanol precipitation, the product was taken up in 10μl water. 0.5 μl were removed for counting to calculate the recovery. 2μl 10×ligase buffer, 2 μl 200 mM DTT, 2 μl 1 mM spermidine, 1 μl 10 mMATP, 3 μl water and 0.5 units kinase were added to the rest (totalvolume 20 μl). The reaction was left at 37° C. for 1 hour and stopped byheating at 90° C. for 2 minutes. The final product was ethanolprecipitated.

Construction of plasmids expressing novel, modified interferons

This section lists and identifies the vectors employed in the cloning ofthe synthetic DNA fragments (Chart 2) into the IFN-β coding region, therestriction enzyme sites* used for the insertion, and the rationale forthe construction. The positions of these sites* are shown relative tothe complete coding nucleotide sequences of the group III novel IFNgenes (Chart 3). The IFN-β (or novel IFN) coding region is shown as aheavy line and would be translated from left to right. The vectorsequences between the BamHI site and the EcoRI site are the same asthose in pAT153 (equivalent to pBR322 with a 705bp HaeII fragmentdeleted-nucleotides 1,646-2,351 on the map). The E. coli trp promoter(Chart 4) lies between the EcoRI site and ClaI site.

1. IFNX423 IFN-β[β⁸²⁻¹⁰⁵ →α₁ ⁸⁰⁻¹⁰³ ][Leu⁸⁴ →Pro]

This novel, modified IFN was designed to determine the effect(s) ofreplacing the computer-predicted C β-helix of IFN-β with that of IFN-α₁on antiviral, antiproliferative and immunostimulating activities(Sternberg and Cohen, Int. J. Biol. Macromol., 4, 137, 1982). Also, whatwould be the effect of shortening this α-helix by introducing a prolineat residue 84?

Starting vector: pGC206. This vector expresses IFN-β from a part natural(amino acids 1-46) and part synthetic IFN-β gene (amino acids 47-166 and(Chart 3c). It was constructed by replacing the 257bp E.coRI-PvuIIfragment of pMN47 with the equivalent fragment from pl/24C. pMN47contains an entirely synthetic IFN-β gene (Chart 3c) inserted betweenthe ClaI and BamHI sites of pl/24C, the plasmid containing the entirelynatural IFN-β gene. (pl/24C is identical to pl/24 (UK Patent ApplicationNo. GB 2 068 970A) except for the underlined sequences in Chart 4).##STR1## A synthetic oligonucleotide (Chart 2a) was inserted between theNruI* and SacII* sites of pGC206 to give the nucleotide sequence shownin Chart 3a. The resultant IFNX423 gene is expressed from plasmidpGC215.

2. IFNX429 IFN-β[β⁸²⁻¹⁰⁵ --α₁ ⁸⁰⁻¹⁰³ ]

The rationale and starting vector was the same as for IFNX423 above. InIFNX429 the predicted α-helical region (82-105) was not shortened by theintroduction of proline at amino acid residue 84. A syntheticoligonucleotide (Chart 2b) was inserted between the NruI* and SacII*sites of pGC206 to give the nucleotide sequence shown in Chart 3b. Theresultant IFNX429 gene is expressed from plasmid pGC2154.

3. IFNX405 IFN-β[¹⁰³⁻¹¹² →α₁ ¹⁰¹⁻¹¹⁰ ]

This novel, modified IFN was designed to determine the effect(s) ofreplacing a relatively non-conserved region (IFN-β cf. IFN-α₁) onantiviral, antiproliferative and immunostimulating activities.

Starting vector: pl/24C This vector expresses mature IFN-β and isidentical to pl/24 except for the ribosome binding site sequenceunderlined in Chart 4. ##STR2## A synthetic oligonucleotide (Chart 2c)was inserted between the MboII* sites (cut sites equivalent to aminoacids 102 and 113) of pl/24C to give the nucleotide sequence shown inChart 3d. The resultant IFNX405 is expressed from plasmid pXX405.

Expression of novel, modified IFNs in Escherichia coli

All the above mentioned plasmids were grown i E. coli HB101 in thepresence of a low level of tryptophan to an OD₆₀₀ of 0.5, then inducedfor IFN synthesis. The medium (200 ml) contained: M9 salts, 0.5%glucose, 0.1 mM CaCl₂, 0.5% Casamino acids, 1 mM MgSO₄, 0.1 mg/mlvitamin B₁, 2.5 μg/ml tryptophan and 100 μg/ml carbenecillin.

200 ml of medium was inoculated with 2-4 ml of an overnight culture ofeach clone (in the host E. coli HB101) grown in the above medium exceptfor the presence of 42.5 μg/ml tryptophan, and grown at 37+ C. withvigorous aeration. At OD₆₀₀ of 0.5, indole acrylic acid, the inducer ofthe E. coli trp promoter and therefore also of IFN synthesis, was addedto 20 μg/ml. At 4-5 hours after induction 3 ml of culture was withdrawn(OD₆₀₀ =0.75-1.2 range) and split as follows: 1 ml was for estimation oftotal "solubilized" IFN antiviral or antiproliferative activity (theactivity regained after a denaturation/renaturation cycle); and 1 ml wasfor display of the total accumulated E. coli proteins plus IFN in apolyacrylamide gel.

(a) Estimation of TOTAL "solubilized" IFN antiviral activity

For recovery of TOTAL "solubilized" IFN antiviral activity, the pelletswere vortexed in 20 μl "lysis buffer" per 0.1 OD₆₀₀ per ml of culture.("Lysis buffer" is 5M urea, 30 mM NaCl, 50 mM Tris-HCl pH7.5, 1% SDS, 1%2-mercaptoethanol, 1% HSA). The mixture was heated for 2-3 minutes at90° C., frozen at -70° C. for 15 minutes, thawed and centrifuged at 17Krpm for 20 minutes. The supernatant was diluted in 1 log steps to 1:10⁵,and appropriate dilutions immediately assayed for IFN antiviral activityby monitoring the protection conferred on Vero cells against thecytopathic effect (cpe) of EMC virus in an in vitro micro-plate assaysystem (e.g. see Dahl and Degre, Acta. Path. Microbiol. Scan., 1380,863, 1972). The diluent was 50 mM Tris-HCl pH7.5, 30 mM NaCl, 1% humanserum albumin (HSA).

(b) Polyacrylamide gel electrophoresis of total polypeptides

Cells from 1 ml of culture were mixed with 10 μl per 0.1 OD₆₀₀ per ml offinal sample buffer: 5 M urea, 1% SDS, 1% 2-mercaptoethanol, 50 mMTris-HCl pH7.5, 30 mM NaCl and 0.05% bromophenol blue. The mixture washeated at 90° C. for 5 minutes, centrifuged for 10 minutes and 5-7 μlloaded on a 15% acrylamide/0.4% bisacrylamide "Laemmli" gel.Electrophoresis was at 70 V for 18 hours. The gel was fixed and stainedwith Coomassie brilliant blue, then dried and photographed.

(c) Antiproliferative assays of modified, novel interferons

Antiproliferative activity was assessed by the ability of the IFN toinhibit the replication of Daudi lymphoblastoid cells (Horoszewicz etal, Science, 206, 1091, 1979). Daudi cells (in log phase) were culturedfor 6 days in 96 well plates in the presence of various dilutions ofinterferon. The phenol red indicator in the medium changes from red toyellow (more acid) with progressive cell growth. Liquid paraffin wasadded to prevent pH change on exposure to the atmosphere and the pHchange in the medium measured colorimetrically on a Dynatech platereader. Interferon inhibition of cell growth is reflected by acorresponding reduction in the colour change.

Comparison of IFN protein expression, antiviral activity andantiproliferative activity in bacterial extracts

Table 1 sets out the expression levels and antiproliferative andantiviral activities of the group III novel, modified IFNs in crudebacterial extracts. A range of activities may be given, reflectingnatural variation in a biological system or assay. The activity quotedis that which is regained after SDS/urea/mercaptoethanol treatment, bydiluting the extract in 1% human serum albumin, as above.

                  TABLE 1                                                         ______________________________________                                                                            Daudi cell                                                                    Anti-                                                      Expression                                                                              EMC/Vero proliferative                                              (% of total                                                                             Antiviral                                                                              activity                                  Novel,   IFNX    cell      activity Units/ml at                               modified IFN                                                                           No.     protein)  IU/L/OD.sub.600                                                                        IC.sub.50 *                               ______________________________________                                        β[ 82-105 →                                                                423     >20       1.3-3.6 × 10.sup.7                                                               1.3 × 10.sup.3                      α.sub.1 80-103 ]                                                        [Leu84→ Pro]                                                           β[ 82-105 →                                                                429     >20       2 × 10.sup.8                                                                     n.d.                                      α.sub.1.sup.80-103 ]                                                    IFN-β                                                                             --       10       0.5-2 × 10.sup.8                                                                 3.4 × 10.sup.3                      control                                                                       ______________________________________                                         n.d. = not done                                                               *Units/ml at IC.sub.50 = dilution of sample assayed for antiviral activit     giving 50% inhibition of cell growth.                                    

It may be seen in Table 1 that for the control, IFN-β, antiviral (AV)and antiproliferative (AP) activity vary over not more than a 4-foldrange (>20 experiments). While the in vitro antiproliferative activityof IFNX423 is not significantly different from IFN-β, the antiviralactivity is lower (˜3 to 30-fold). This may be due in part to theproline at amino acid residue 84, since IFNX429, which is identical toIFNX423 except for leucine at residue 84, displays similar antiviralactivity to IFN-β. Therefore, shortening the predicted "C" α-helix bythe introduction of a proline may adversely affect in vitro antiviralactivity. In conclusion, IFNX423 and IFNX429 are examples of novel,modified IFNs which have lost part of their antiviral activities.

Biological Properties of IFNX405 1. Methods

The expressed proteins were extracted from E. coli with the aid ofsodium dodecyl sulphate (SDS) and purified by chromatography on AcA44.The IFNs had estimated purity of 70-90% based on polyacrylamide gelelectrophoretic (PAGE) analysis.

The novel interferons were tested to determine their specific antiviral,antiproliferative and immunomodulatory activities. The following assaysystems were employed:

(i) Antiviral assay

(a) Cytopathic effect (CPE) assay with encephalomyocarditis (EMC) virus.This is a standard assay which measures the ability of interferon toprotect cell monolayers against the cytopathic effect of EMC virus. Thecell lines used were: Vero (African Green Monkey epithelial), WISH(amnion epithelial), MRC-5 (foetal lung fibroblast) and 17-1 (foetallung fibroblast). Cell monolayers were established in 96 wellflat-bottomed microtitre plates in DMEM medium containing 2% donor calfserum plus glutamine and antibiotics. Serial 1 in 2 dilutions ofinterferon were incubated with the cells at 37° for 18-24 hours, thesupernatant discarded and an appropriate challenge dose of EMC virus inmedium added. After incubation at 37° for a further 24 hours, thesupernatants were discarded, the monolayers fixed with formol/saline andstained with crystal violet. The plates were read visually to establishthe dilution of interferon giving 50% inhibition of the cytopathiceffect.

(b) Plaque reduction assay--using Herpes simplex type 2 (HSV-2) viruswith Vero (monkey) Chang (human) and MDBK (bovine cells). Confluentmonolayers of cells were established in 96 well flat-bottomed microtitreplates. After incubation at 37° for 18 hours with dilutions ofinterferons, the cells were challenged with an appropriate number ofplaque forming units of virus, overlaid with medium containing 0.5%carboxymethyl cellulose and incubated at 37° for 24 hours. Afterfixation and staining the plaques were counted microscopically and thecounts expressed as a percentage of the mean maximum plaque counts inuntreated control wells. Interferon titres are the reciprocal dilutionsgiving 50% reduction in plaque number/well.

(ii) Antiproliferative assay

Daudi cells in Dulbecco's Modified Eagles Medium (DMEM) were seeded at2×10⁵ /ml (200 μl) in 96 well tissue culture plates. Interferons wereadded at the time of seeding and cells incubated at 37+ in a humidified5% CO₂ atmosphere. After 22 hours, tritiated thymidine was added and thecells incubated for a further 2 hours after which they were harvested ona Flow cell harvester washed and treated with 5% trichloroacetic acid.Acid insoluble radioactivity was counted and inhibition of thymidineincorporation was taken as a measure of the antiproliferative activityof interferon.

(iii) Immunomodulatory assay (Natural Killer (NK) Cell Activity

Buffy coat cells separated from human peripheral blood by Ficoll/Hypaquesedimentation were suspended in supplemented RPMI 1640 medium andincubated overnight at 37° with interferon dilutions. After washing toremove interferon, these effect or cells were incubated at 37° for afurther 4 hours with ⁵¹ Cr-labelled K562 cells at effector to targetcell ratios of 20:1 or 10:1. (K562 is a human tumour-derived cell line).After centrifugation an aliquot of the supernatant was removed formeasurement of released radioactivity. Maximum ⁵¹ Cr release wasobtained by repeated freeze-thawing of a target cell suspension and abackground control obtained by measurement of ⁵¹ Cr release from targetcells incubated without effector cells. Results were expressed aspercentage specific ⁵¹ Cr release: ##EQU1##

2. Results

(i) Antiviral activities

(a) CPE assay--EMC virus

Table 2 lists the assay means for the hybrid IFNX405 and therecombinant-derived IFN-β measured against EMC virus in Vero and thefour human cell lines. The activities are expressed in units/mg protein.

From the individual interferon means in different cell types containedin Table 2 and from the summary pooled data across all cell types it isseen that IFNX405 has activity very similar to that of IFN-β in thedifferent cell lines.

                  TABLE 2                                                         ______________________________________                                        Antiviral activities of IFN-β and IFNX405 against                        encephalomyocarditis virus (IFN units/mg protein)                             ______________________________________                                        Mean activities in each cell line                                                                       CELL                                                PREPA-                    LINE                                                RATION  Vero     Chang    WISH   MRC-5  17-1                                  ______________________________________                                        IFN-β x                                                                          1.5 × 10.sup.5                                                                   5.2 × 10.sup.5                                                                   8.4 × 10.sup.5                                                                 1.5 × 10.sup.5                                                                 7.1 × 10.sup.4                  IFNX405 x                                                                             1.4 × 10.sup.5                                                                   2.6 × 10.sup.5                                                                   1.0 × 10.sup.6                                                                 1.5 × 10.sup.5                                                                 5.5 × 10.sup.4                  ______________________________________                                                                  95% CONFIDENCE                                      PREPARATION POOLED MEAN   LIMITS (u/mg)                                       ______________________________________                                        IFN-β  2.4 × 10.sup.5 u/mg                                                                   1.5-3.9 × 10.sup.5                            IFNX405     1.4 × 10.sup.5 u/mg                                                                   0.8-2.3 × 10.sup.5                            ______________________________________                                         (-x calculated based upon 3-5 assays)                                    

For comparative purposes, the observed activities (in units/ml) ofpreparations of fibroblast IFN-β and leucocyte IFN-α are shown in Table3. These natural interferons were not available in purified form andwere used in the assays in dilute solutions containing large amounts ofnon-interferon protein. Thus, results with natural IFN-β and IFN-αcannot be quoted in units/mg and the results in Table 3 are not directlycomparable with those of Table 2. Nevertheless, it can be seen that theactivity of both natural interferons is sustained across the five celllines within an interferon class with the exception that WISH cellsappear slightly more sensitive to both IFN-β and IFN-α.

                  TABLE 3                                                         ______________________________________                                        Relative antiviral activities of natural interferon                           preparations against encephalomyocarditis virus in vero and                   human cell lines                                                              Interferon units/ml                                                                                     CELL                                                PREPA-                    LINE                                                RATION  Vero     Chang    WISH   MRC-5  17-1                                  ______________________________________                                        Fibroblast-                                                                           3.6 × 10.sup.4                                                                   5.6 × 10.sup.4                                                                   1.3 × 10.sup.5                                                                 7.8 × 10.sup.4                                                                 6.8 × 10.sup.4                  derived β x                                                              Leucocyte-                                                                            2.5 × 10.sup.2                                                                   1.5 × 10.sup.2                                                                   1.3 × 10.sup.3                                                                 80     80                                    derived                                                                       IFN-α x                                                                 ______________________________________                                    

(b) Plaque reduction assays HSV-2

Similar estimates of antiviral activities obtained with HSV-2 by meansof plaque reduction assays are given in Table 4. In this case theexperiments were confined to the human Chang cells, primate Vero cellson bovine MDBK cells. IFNX405 has similar activity to IFN-β in Chang andVero but reduced activity in MDBK.

The pattern of natural IFN-β and IFN-α against HSV-2 in these 3 celllines is shown in Table 5, again expressed as units/ml rather than asspecific activity as a result of impure IFNs. In contrast to somereported results from other laboratories, IFN-β reacts reasonably wellwith our MDBK cell line, producing antiviral activity at about the samedilution as Vero or Chang cells. On the other hand, the IFN-α standardreacted substantially better with MDBK cells than with either Vero orChang cells.

                  TABLE 4                                                         ______________________________________                                        Antiviral activities of IFN-β and IFNX405 against HSV-2                  determined by plaque reduction assay                                          Interferon units/mg protein                                                                          CELL LINE                                              PREPARATION   Vero     Chang        MDBK                                      ______________________________________                                        IFN-β x  1.2 × 10.sup.5                                                                   4.7 × 10.sup.5                                                                       2.5 × 10.sup.5                      IFNX405 x     5.4 × 10.sup.4                                                                   2.0 × 10.sup.5                                                                       1.8 × 10.sup.4                      ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        Relative antiviral activity of natural interferons against                    HSV-2 in monkey, human and bovine cells determined by plaque                  reduction assays                                                              Interferon units/ml                                                                                  CELL LINE                                              PREPARATION  Vero      Chang       MDBK                                       ______________________________________                                        Fibroblast-derived                                                                         2.6 × 10.sup.4                                                                    9.3 × 10.sup.4                                                                      1.9 × 10.sup.4                       IFN-β x                                                                  Leucocyte-derived                                                                          59        90          6.8 × 10.sup.3                       IFN-α x                                                                 ______________________________________                                    

Summarizing the results of antiviral activity with RNA and DNA virusesin relevant cell types, Table 6 lists the activities of the recombinantand natural interferons against EMC and HSV-2 in Chang and Vero cells(data from Tables 2-5). There is no indication from these results ofpreferential activity of IFNX405 against one or other of the 2 virustypes. The results from the 2 sets of assays are remarkably similar andare not significantly different. Thus the pooled mean antiviral activityagainst EMC virus shown in the analysis of variance to Table 2 isequally valid as an estimate of antiherpes activity and can be used asan overall indicator of specific antiviral activity of IFNX405.

                  TABLE 6                                                         ______________________________________                                        Relative antiviral activity against encephalomyocarditis                      virus and HSV-2 for IFN-β and IFNX405 assayed in human and               monkey cells                                                                  Interferons (unit/mg protein)                                                          Pooled mean activity                                                                          Pooled mean activity                                 IFN      EMC virus       HSV-2                                                Preparation                                                                            (from Table 1 analysis)                                                                       Vero and Chang cells                                 ______________________________________                                        IFN-β                                                                             2.4 × 10.sup.5                                                                          3.5 × 10.sup.5                                 IFNX405  1.4 × 10.sup.5                                                                          1.3 × 10.sup.5                                 ______________________________________                                    

(c) Comparative antiviral data with an atypical Chang cell line

One line of Chang conjunctival cells maintained in high passage (approx.X 160) has undergone a mutational change such that it is approximately 3times more sensitive to IFN-β than the normal low passage Chang cellswhich we have used in routine assays. At the same time, the atypicalhigh passage Chang cells recognize and respond to IFN-α with a 100-foldincrease in sensitivity compared to the parental low passage Changcells. Comparative ratios of antiviral activity in high and low passageChang cells can therefore be used to indicate a degree of α-likeproperty in a particular recombinant.

The results of profiling the recombinant IFNX405 in this way is shown inTable 7.

(ii) Antiproliferative activity

IFN-β and IFNX405 were assayed for growth inhibitory activity againstDaudi lymphoblastoid cells in at least 4 replicate assays. The meanresults of these assays are given in Table 8, activities being expressedas the potein concentration required to prouce a 50% inhibition ofmaximum thymidine incorporation in untreated control cells (InhibitoryDose₅₀). IFNX405 has lost activity, and although the loss is slight, itis significant as shown by analysis of variance.

                  TABLE 7                                                         ______________________________________                                        Antiviral activities of IFN-β IFNX405 in a typical Chang                 cells compared with natural and interferons                                          Chang.sup.A                                                                              Chang (Routine                                                                             Ratio                                                 (High passage)                                                                           low passage) ChA/Ch                                         ______________________________________                                        Units/mg                                                                      IFN-β                                                                             1.6 × 10.sup.6                                                                       5.2 × 10.sup.5                                                                       3                                          IFNX405  3.0 × 10.sup.5                                                                       2.6 × 10.sup.5                                                                       1                                          Units/ml                                                                      Fibroblast                                                                             1.7 × 10.sup.5                                                                       5.6 × 10.sup.4                                                                       3                                          IFN-β                                                                    Leucocyte                                                                              3.4 × 10.sup.4                                                                       1.5 × 10.sup.2                                                                       226                                        IFN-α                                                                   ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        Antiproliferative activity of IFN-β and IFNX405 assayed in               Daudi human lymphoblastoid cells                                              Inhibitory Dose.sub.50 (μg/ml)                                             PREPA-   No. of replicate                                                                          Corrected  95% Confidence                                RATION   assays (n)  Mean ID.sub.50                                                                           Limits                                        ______________________________________                                        IFN-β                                                                             4           3.8        1.5-9.8                                       IFNX405  6           7.1         3.2-15.5                                     ______________________________________                                    

(iii) Immunomodulatory activity-NK assay

IFN-β and IFNX405 were also repeatedly assayed for ability to enhancenatural killer (NK) cell activity, a total of 9-11 assays contributingto the results which are shown in Table 9. In a similar fashion to theantiproliferative activity, the specific NK stimulating activity isexpressed as the protein dose concentration producing a 50% effect(Stimulating Dose ₅₀).

IFNX405 has reduced NK stimulating activity being about 4-fold lessactive than IFN-β parent. This difference is significant as shown in theanalysis of variance.

                  TABLE 9                                                         ______________________________________                                        Immunostimulant activities of IFN-β and IFNX405 assayed with             human NK cells                                                                PREPA-   No. of replicate                                                                          Corrected  95% Confidence                                RATION   assays (n)  Mean SD.sub.50                                                                           Limits                                        ______________________________________                                        IFN-β                                                                             11           3.4       2.1-5.4                                       IFNX405   9          14.5        8.5-24.5                                     ______________________________________                                    

3. Conclusions

Mean specific activities for the antiviral, antiproliferative andimmunodulatory properties of each interferon are summarized in Table 10.(It should be noted that activity varies directly with the figures takenfrom antiviral assays but inversely with the figures quoted from ID₅₀and SD₅₀ assays). For convenience these results have been indexedrelative to the IFN-β parent in the lower half of Table 10. From thisanalysis it may be seen that IFNX405 has identical antiviral activity toIFN-β but has lost a small part of its antiproliferative andimmunostimulating properties.

                  TABLE 10                                                        ______________________________________                                        Comparative summary of biological data for recombinant and                    natural interferons                                                                               Specific    Specific                                              Specific    antiproliferative                                                                         immunostimulant                               PREPA-  antiviral   activity    activity                                      RATION  activity (u/mg)                                                                           (ID.sub.50 μg/ml.sup.-1)                                                               (SD.sub.50 μg/ml.sup.-1)                   ______________________________________                                        IFN-β                                                                            2.4 × 10.sup.5                                                                      3.8         3.4                                           IFNX405 1.4 × 10.sup.5                                                                      7.1         14.5                                          Indexed results (IFN-β = 100)                                            IFN-β                                                                            100         100         100                                           IFNX405 100 (58)    54          23                                            ______________________________________                                         Figures in brackets indicate actual calculated index where it is not          significantly different from 100. In all other cases, differences from 10     are significant.                                                         

Pharmaceutical formulation and administration

The novel, modified interferons of the present invention can beformulated by methods well known for pharmaceutical compositions,wherein the active interferon is combined in admixture with apharmaceutically acceptable carrier substance, the nature of whichdepends on the particular mode of administration being used. Remington'sPharmaceutical Sciences by E. W. Martin, hereby incorporated byreference, describes compositions and formulations suitable for deliveryof the interferons of the present invention. For instance, parenteralformulations are usually injectable fluids that use physiologicallyacceptable fluids such as saline, balanced salt solutions, or the likeas a vehicle. Oral formulations may be solid, e.g. tablet or capsule, orliquid solutions or suspensions.

The novel, modified interferons of the invention may be administered tohumans or other animals on whose cells they are effective in variousways such as orally, intravenously, intramuscularly, intraperitoneally,intranasally, intradermally or subcutaneously. Administration of theinterferon composition is indicated for patients with malignancies orneoplasms, whether or not immunosuppressed, or in patients requiringimmunomodulation, or antiviral treatment. Dosage and dose rates mayparallel those employed in conventional therapy with naturally occurringinterferons--approximately 10⁵ to 10⁸ units daily. Dosages significantlyabove or below these levels may be indicated in long term administrationor during acute short term treatment. A novel, modified interferon maybe combined with other treatments or used in association with otherchemotherapeutic or chemopreventive agents for providing therapy againstthe above mentioned diseases and conditions, or other conditions againstwhich it is effective.

Modifications of the above described modes for carrying out theinvention such as, without limitation, use of alternative vectors,alternative expression control systems, and alternative hostmicro-organisms and other therapeutic or related uses of the novelinterferons, that are obvious to those of ordinary skill in thebiotechnology, pharmaceutical, medical and/or related fields areintended to be within the scope of the following claims.

                                      CHART 2a                                    __________________________________________________________________________    Chemically synthesized sequence for IFNX423                                   __________________________________________________________________________     ##STR3##                                                                     GCACCGAACTGTACCAGCAACTGAACGACCTGGAAGCATGTGTTATGCAGGAACTGGAAA                  CGTGGCTTGACATGGTCGTTGACTTGCTGGACCTTCGTACACAATACGTCCTTGACCTTT                   ##STR4##                                                                     __________________________________________________________________________

                                      CHART 2b                                    __________________________________________________________________________    Chemically synthesized sequence for IFNX423                                   __________________________________________________________________________     ##STR5##                                                                     GCACCGAACTGTACCAGCAACTGAACGACCTGGAAGCATGTGTTATGCAGGAACTGGAAA                  CGTGGCTTGACATGGTCGTTGACTTGCTGGACCTTCGTACACAATACGTCCTTGACCTTT                   ##STR6##                                                                     __________________________________________________________________________

                  Chart 2c                                                        ______________________________________                                        Chemically synthesized sequence for IFNX405                                   ______________________________________                                         ##STR7##                                                                     ______________________________________                                    

                                      CHART 3a                                    __________________________________________________________________________    IFNX423                                                                        ##STR8##                                                                     __________________________________________________________________________     ##STR9##                                                                      ##STR10##                                                                     ##STR11##                                                                     ##STR12##                                                                     ##STR13##                                                                     ##STR14##                                                                     ##STR15##                                                                     ##STR16##                                                                     ##STR17##                                                                     ##STR18##                                                                     ##STR19##                                                                     ##STR20##                                                                     ##STR21##                                                                     ##STR22##                                                                     ##STR23##                                                                     ##STR24##                                                                    __________________________________________________________________________

                                      CHART 3b                                    __________________________________________________________________________    IFNX429                                                                        ##STR25##                                                                    __________________________________________________________________________     ##STR26##                                                                     ##STR27##                                                                     ##STR28##                                                                     ##STR29##                                                                     ##STR30##                                                                     ##STR31##                                                                     ##STR32##                                                                     ##STR33##                                                                     ##STR34##                                                                     ##STR35##                                                                     ##STR36##                                                                     ##STR37##                                                                     ##STR38##                                                                     ##STR39##                                                                     ##STR40##                                                                     ##STR41##                                                                    __________________________________________________________________________

                                      CHART 3c                                    __________________________________________________________________________    Synthetic IFN-β gene                                                     __________________________________________________________________________     ##STR42##                                                                     ##STR43##                                                                     ##STR44##                                                                     ##STR45##                                                                     ##STR46##                                                                     ##STR47##                                                                     ##STR48##                                                                     ##STR49##                                                                     ##STR50##                                                                     ##STR51##                                                                     ##STR52##                                                                     ##STR53##                                                                     ##STR54##                                                                     ##STR55##                                                                     ##STR56##                                                                    QKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLE                         EKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYF                          ##STR57##                                                                    __________________________________________________________________________

                                      CHART 3d                                    __________________________________________________________________________    IFNX405                                                                        ##STR58##                                                                    __________________________________________________________________________     ##STR59##                                                                     ##STR60##                                                                     ##STR61##                                                                     ##STR62##                                                                     ##STR63##                                                                     ##STR64##                                                                     ##STR65##                                                                     ##STR66##                                                                     ##STR67##                                                                     ##STR68##                                                                     ##STR69##                                                                     ##STR70##                                                                     ##STR71##                                                                     ##STR72##                                                                     ##STR73##                                                                     ##STR74##                                                                    __________________________________________________________________________

                                      CHART 4                                     __________________________________________________________________________    Nucleotide sequence of trp promoter region of IFN-β expression           plasmid pl-24/C                                                               __________________________________________________________________________     ##STR75##                                                                     ##STR76##                                                                    __________________________________________________________________________

We claim:
 1. A modified beta interferon comprising a beta interferonwherein amino acids 82 to 105 of said beta interferon are replaced byamino acids 80 to 103 of alpha 1 interferon.
 2. A modified betainterferon comprising a beta interferon wherein amino acids 82 to 105 ofsaid beta interferon are replaced by amino acids 80 to 103 of alpha 1interferon with the exception that the leucine which is located atposition 84 of natural beta interferon is replaced by proline.
 3. Amodified beta interferon comprising a beta interferon wherein aminoacids 103 to 112 of said beta interferon are replaced by amino acids 101to 110 of alpha 1 interferon.
 4. A pharmaceutical composition for use inthe treatment of viral infections in an animal comprising an effectivetherapeutic amount of the modified beta interferon of claim 1 admixedwith a pharmaceutically acceptable carrier.
 5. A pharmaceuticalcomposition for use in the treatment of viral infections in an animalcomprising an effective therapeutic amount of the modified betainterferon of claim 2 admixed with a pharmaceutically acceptablecarrier.
 6. A pharmaceutical composition for use in the treatment ofviral infections, or neoplastic disease or for stimulating the immunesystem in an animal comprising an effective therapeutic amount of themodified beta interferon of claim 3 admixed with a pharmaceuticallyacceptable carrier.
 7. A method of treating viral infections in ananimal in need of such treatment comprising the administration of aneffective amount of the modified beta interferon of claim
 3. 8. A methodof regulating cell growth in an animal in need of such treatmentcomprising the administration of an effective amount of the modifiedbeta interferon of claim
 3. 9. A method of regulating the immune systemin an animal in need of such treatment comprising the administration ofan effective amount of the modified beta interferon of claim 3.